Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha.
The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha.
Dai
R M
RM
Intramural Research Support Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Chen
E
E
Longo
D L
DL
Gorbea
C M
CM
Li
C C
CC
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D006868:Hydrolysis; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D014452:Ubiquitins / Q000378:metabolism*; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein